Genetic determinants of mycobacterial susceptibility and pathogenesis

When bacteria enter a host, such as a person or other animal, they are typically detected by the immune system of the host. The immune response then attempts to kill the invading bacteria. In most cases the immune response is successful. In some cases, though, the immune response fails to kill the invader, and the invading bacteria can become established and grow in the host. When this happens, disease is the result. In many diseases, including tuberculosis, the immune response is not shut off by the bacteria; instead, it is active, but ineffective at killing the invader. In these cases, most of the damage caused by the disease is not caused by the bacteria directly; rather, the damage is caused by this ineffective immune response. The goal of this proposal is to understand how disease-causing bacteria can escape the immune response and become established in the host, and how immune responses, which are supposed to just kill invaders, can instead come to cause so much damage to the host. In order to examine these issues, we developed a new system for the study of infection. In this system, a bacterium called Mycobacterium marinum, which is closely related to the bacterium that causes tuberculosis, is injected into a fruit-fly (Drosophila melanogaster). This system is very useful because much of the immune response to bacteria is similar between flies and higher animals, such as humans or cattle. At the same time, we know a great deal about flies, and have excellent tools for working with them, so that we can easily find flies with mutations that make them more- or less-efficient at fighting this infection, or mutations that make the infection less damaging to the fly. Using these techniques, we can find genes that are important for fighting infections or that cause the damage due to the immune response. These genes are difficult to find in higher organisms, such as mice, because the process is very time-consuming and involves many animals. For example, in order to do the experiments in this proposal in mice would take more than ten years and more than 10,000 mice. The fly allows us to study these genes rapidly and cheaply, without the ethical issues inherent in the use of mice or other mammals.

My laboratory is interested in the mechanisms by which hosts affect the course of bacterial infections. This includes immune effects, both in terms of eradicating the pathogen and in terms of causing unintended damage to the host. To address these issues, I developed a model system in which Drosophila melanogaster is infected with the broad-spectrum pathogen Mycobacterium marinum, a close relative of the M. tuberculosis complex. This system allows us to find host genes that contribute to the biology of infection in a rapid and unbiased way without using vertebrate animals. I propose to continue and expand a genetic screen I began as a post-doc. The initial screen was for Drosophila mutants that died more quickly or more slowly after M. marinum infection. This screen will be continued and coupled with a second screen for mutants that are unable to fight a non-pathogenic mycobacterial species, M. smegmatis. By screening all mutants for susceptibility to both bacteria we can find genes that contribute to bacterial pathogenesis, help the host kill mycobacteria, or are circumvented by pathogens in order to cause disease. This work has three specific aims: Aim 1. Continuation and expansion of the already-begun genetic screen. We will screen at least 5000 mutant Drosophila lines for changes in time-to-death when infected with M. marinum and susceptibility to M. smegmatis. Aim 2. Secondary screens and basic characterization of many mutants. All mutants from the primary screen will be subjected to a battery of secondary screens to help us organize them into functional classes. Aim 3. Deeper characterization of at least one specific mutant. At least one mutant will be characterized in detail. This work is important and exciting because it affords us an unbiased view of what host processes are important to disease susceptibility and the progress of pathogenesis, important questions that are difficult to answer using more conventional infection models.